ABSTRACT Neurotensin (NT) is a tridecapeptide localized to specialized enteroendocrine (EE) cells predominantly in the small bowel. The most potent stimulus for NT release is the ingestion of dietary fats. NT facilitates free fatty acid (FFA) absorption in the proximal intestine, stimulates growth of colorectal, pancreatic and breast cancers that have the high affinity NT receptor 1 (NTR1), and contributes to lipid metabolism and glucose control although its precise role in these processes has not been delineated. Recently, a large population study identified a significant association of increased fasting pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) levels with the development of diabetes, increased risk of cardiovascular disease and mortality, and increased risk of breast cancer in women. Together, these findings identify an important role for NT in lipid metabolism and, moreover, links increased NT levels to various metabolic diseases and increased morbidity and mortality. Epidemiological evidence clearly shows direct linkage between overnutrition and obesity; however, the molecular mechanisms linking adiposity to overnutrition remain unknown. In exciting recent findings, we demonstrate that NT deficiency (using an NT knockout mouse model) protects against obesity, insulin resistance and non-alcoholic fatty liver disease (NAFLD) associated with high fat consumption; we further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates FFA absorption through a mechanism involving NTR1 and NTR3/sortilin. Conversely, the overexpression of NT in Drosophila midgut EE cells increases lipid accumulation in the midgut, fat body and oenocytes (hepatocyte-like cells). Remarkably, in humans, we show that increased levels of pro-NT strongly predict new onset obesity in a graded manner, which is independent of body mass index (BMI) and insulin resistance. Therefore, the central hypothesis for our current proposal is that FFA-mediated NT release by EE cells, through a cross-talk mechanism involving AMPK activation, mTOR inhibition, and ERK1/2 activation, promotes intestinal absorption of FFAs acting through NTR1 and/or NTR3 and the inhibition of intestinal AMPK. Moreover, we speculate that the overconsumption of dietary fats, which leads to excess NT secretion, results in obesity (from continued fat storage) and metabolic disorders (e.g., hepatic steatosis and insulin resistance). To examine our long-term goal of better defining intestinal NT secretion and function, we have assembled a multidisciplinary and highly collaborative team with defined expertise in NT physiology and function; metabolism and systems biochemistry; Drosophila genetics; and, biostatistics/computational biology. Ultimately, our findings will: i) significantly advance the fields of GI physiology, endocrinology and metabolism; ii) change existing paradigms regarding the systemic effects of NT; and, iii) revolutionize our concept of gut hormones and their role in obesity and metabolic diseases.